Hiseq 2500 miseq system

Illumina sequencing analysis of exosomal miRNAs was performed by Novogene using RNA extracted from equal amounts of serum exosomes collected from patients before receiving neoadjuvant chemotherapy, after receiving one cycle of neoadjuvant chemotherapy and after receiving four cycles of neoadjuvant chemotherapy. All small RNAs comprising 15–52 nucleotides were selected and sequenced using the HiSeq™2500/MiSeq system (Illumina) in accordance with the manufacturer’s protocol. Original counts were standardized using the pruned mean mol/L value method, and the results were analyzed using the Bioconductor package “edgeR” to identify the differentially expressed miRNAs for the three types of exosomes.

Mid-vegetative alfalfa seedlings subjected to normal or Pi deficiency treatment for 20 days were used for sequencing analysis. Sequencing of small RNA transcriptome data was conducted as previously described (Li et al., 2018 (link)). Then, the small RNA transcriptome data were downloaded from the GEO database (NCBI accession number: SRP110842). The mRNA transcriptome data were obtained from the same root sample as the small RNA data. The sequencing and analysis were conducted by Shanghai Majorbio Technology Co Ltd.¹ Briefly, total RNA was extracted, and its integrity was validated before further analysis. A library was constructed using RNA for each sample. Then, the cDNA libraries were sequenced on the Illumina Hiseq2500/Miseq system (2 × 150 bp), and the obtained sequences were uploaded to the NCBI database (accession number: SRP133551).